WO2022090601A1

WO2022090601A1 - Industrial-scale process for obtaining a concentrated extract of biopesticidal compounds from microalgae and/or cyanobacteria, resulting extract, and use thereof

Info

Publication number: WO2022090601A1
Application number: PCT/ES2021/070775
Authority: WO
Inventors: Elvira NAVARRO LÓPEZ; Emilio Molina Grima; Francisco Gabriel ACIEN FERNÁNDEZ; José María FERNÁNDEZ SEVILLA; María del Carmen CERÓN GARCÍA; Elisa Rojas Crespo; Juan José GARCÍA LÓPEZ; Joaquín POZO DENGRA
Original assignee: Biorizon Biotech, S.L.
Priority date: 2020-10-26
Filing date: 2021-10-26
Publication date: 2022-05-05
Also published as: ES2908029B2; ES2908029A1; EP4238419A1; ES2908029B8

Abstract

The invention relates to a process for the extraction of compounds having inhibitory activity against the growth of phytopathogenic microorganisms from microalgae and/or cyanobacteria biomass, the extract thus obtained, and the use thereof as a biopesticide in agriculture. The process essentially comprises: obtaining a suspension of microalgal and/or cyanobacterial biomass at a given solid concentration; releasing the cellular content of the biomass by high-pressure homogenisation, operated at moderate pressure; extracting the biopesticidal components using a mixture of organic solvent, with a polarity between 0 and 9, and water; and separating the extract containing the biopesticidal components from the residual microalgal and/or cyanobacterial biomass using physical methods.

Description

PROCESS FOR OBTAINING, AT THE INDUSTRIAL LEVEL, AN EXTRACT CONCENTRATED IN BIO-PESTICIDE COMPOUNDS FROM MICROALGAE AND/OR CYANOBACTERIA, EXTRACT SO OBTAINED AND USE THEREOF

DESCRIPTION

TECHNICAL FIELD OF THE INVENTION

The present invention is encompassed in the field of the production of biopesticide products for agriculture.

More specifically, the invention provides a process for extracting compounds with activity that inhibits the growth of phytopathogenic microorganisms from biomass of microalgae and/or cyanobacteria, with application in both extensive and intensive agriculture. Consequently, the invention also relates to the use of the extract obtained by said procedure as a biopesticide in agriculture.

BACKGROUND OF THE INVENTION

The environmental impact of chemically synthesized pesticides, as opposed to biopesticides, has become a major concern, not only because of the chemicals' persistence in the upper soil layer or their leaching into groundwater, but also because of its undesired effects on inhabited areas that these volatilized pesticides can reach, being already relevant, in addition to the environmental impact on flora and fauna, also the toxic effect of exposure to this type of chemical on human health.

For this reason, the use of natural compounds for pest control, also called biopesticides, is a viable, sustainable and effective alternative to the massive use of chemically synthesized pesticides. Biopesticides are products obtained from living microorganisms or natural products, which are used to prevent, repel, destroy or mitigate any pest or agent that causes damage to plant crops, humans or other animals. These pests include insects, animals, unwanted plants, microorganisms (bacteria, fungi, viruses), and phones (proteins that can act as infectious agents) (Chandler et al., “The development, regulation and use of biopesticides for integrated pest management", Phil. Trans. R. Soc. B 12 July 2011 vol. 366 no. 1573 1987-1998 2011; Carlos Hernández and Gamboa-Angulo, “Metabolites from freshwater aquatic microalgae and fungi as potential natural pesticides", Phytochemistry Reviews , 2011).

The potential of microalgae and cyanobacteria, as a source for obtaining various products of great interest to agriculture, has gained relevance in recent decades. The incidence of diseases caused by fungi and phytopathogenic bacteria is an obvious problem that generates great concern. In this sense, microalgae and cyanobacteria have positioned themselves as sources of biopesticide compounds of potential use in agriculture. Both groups of photosynthetic organisms are represented by a great variety of species, adapted to very diverse ecological habitats. To survive in a highly competitive environment, often experiencing large fluctuations in physical and chemical parameters, they have developed adaptive and defensive strategies, including the synthesis of numerous compounds obtained by different metabolic pathways.

Some notable compounds synthesized by microalgae and/or cyanobacteria are: substances with antioxidant activity, such as carotenoids and polyphenols; plant growth promoting compounds, such as phytohormones and sterols; and substances with antimicrobial activity, such as fatty acids or polysaccharides. Many of these metabolites have very specific and complex chemical structures not found among terrestrial organisms, making it difficult to reproduce them by chemical synthesis (Falaise et al., “Antimicrobial Compounds from Eukaryotic Microalgae against Human Pathogens and Diseases in Aquaculture", Mar Drugs.2016 Sep 2;14(9), 2016;Michalak & Chojnacka, “Algal extracts: Technology and advances”, Engineering in Life Sciences, Vol. 14, 2014; Najdenski et al., “Antibacterial and antifungal activities of selected microalgae and cyanobacteria,” Food Science and Technology, Col. 48, 2013; Suárez-Estrella et al., “Biological control of plant pathogens by microorganisms isolated from agroindustrial composts”, Biological Control, 2013).

Thus, thanks to their enormous biodiversity, ease of cultivation, rapid growth and metabolism control, microalgae and cyanobacteria are increasingly used to produce bioactive compounds with different applications, being used in the form of both microalgal extracts and purified metabolites (Elarroussi et al. al, “Microalgae polysaccharides a promising plant growth biostimulant”, Journal of Algal Biomass Utilization, 2016).

Microalgae and cyanobacteria have proven to be effective against a wide variety of phytopathogenic agents, in general bacteria, fungi and nematodes. This biocidal activity is mainly due to the production of hydrolytic enzymes and other compounds that cause disruption of the cytoplasmic membrane, inactivation of enzymes, inhibition of protein synthesis, etc. (Renuka et al., “Microalgae as multi-functional options in modern agriculture: current trends, prospects and challenges”, Biotechnology Advances 36(4), 2018). It has been seen that phenolic compounds, such as flavonoids or coumarins, are the main metabolites produced by microalgae that have a broad spectrum of antimicrobial activity (Pina-Pérez et al., “Antimicrobial potential of macro and microalgae against pathogenic and spoilage microorganisms in food”, food Chemistry, 2017). Other compounds isolated from microalgae that have biocidal properties are terpenes, polysaccharides, pigments such as chlorophyll and carotenoids, and fatty acids, such as propanoic acid and butanoic acid (Hernández-Carlos and Gamboa-Angulo, 2011; Michalak and Chojnacka, 2014).

For example, document CN107135829A describes a brown algae extract capable of reducing the concentration of pesticide residues in plants and trees. Document CN103114003A describes a disinfectant prepared from enzyme complexes from marine organisms, phosphatidyl dehydrogenase, enzymes from marine microalgae and marine pyrrolic alkaloids for the same purpose.

The authors of the invention, after extensive experimental work, have developed a new method for extracting biopesticide compounds for agricultural use from biomass of microalgae and/or cyanobacteria.

The novelty of the developed process is based on the fact that it is an industrial, scalable and achievable process at a reasonable cost, unlike the methods described in the state of the art, which are analytical methods and, as such, pursue complete quantification/extraction. of biopesticides without taking into account their scale or cost.

Thus, for example, in the document by Devi KM et al. (“Antimicrobial activities of freshwater cyanobacterium”, Nostoc sp. from tamdil wetland of mizoram, India: An identification of bioactive compounds by GC-MS. International Journal of Pharmaceutical Sciences and Research, 2016, vol. 7 (3), pages 1179 - 1191, DOI:10.13040/IJPSR.0975-8232.7(3).1179-91) describes an analytical method for extracting compounds from biomass (cyanobacteria), using pure solvents or their mixtures, and testing the extracts once eliminated extracting agent completely.It is not in any case an industrial method, since the amounts of solvent used are very high, it requires a purification stage of the extract by chromatography, as well as the use of pure solvents that extract different compounds, because they have polarities very different from those of the present invention.Furthermore, they inevitably require a solvent evaporation step that is not necessary in this invention.

In the Salem et al. ("Antimicrobial activity of microalgal extracts with emphasize on Nostoc sp". Life Science Journal, 2014, vol. 11 (12), pages 752-758) an analytical method for preparing microalgal extracts using pure acetone and methanol is also described. It also includes a breaking stage by grinding after freezing with liquid nitrogen, as well as the removal of the solvent by evaporation. Again, it is an industrially unviable method that also extracts different compounds based on the different extraction conditions.

In the article by Saber A et al. (Insecticidal prospects of algal and cyanobacterial extracts against the cotton leafworm Spodoptera littoral is. Vie et Milieu 2018, vol. 68 (4), pages 199-212, ISSN 0240-8759) an analytical method of extraction from biomass of microalgae, which uses pure solvents, very long contact times of several hours, very excess amounts of solvent to biomass, and breakage methods only applicable on a laboratory scale.

On the other hand, in the state of the art, microalgae and cyanobacteria are always harvested wet and subsequently dried because, otherwise, the biomass rapidly corrupts. For this reason, the methods described in the state of the art start with dry biomass, which is subjected to humidity just before starting the extraction processes.

The authors of the invention have thus had to overcome many technical difficulties in order to develop a method that allows the extraction of biopesticide compounds from microalgae and/or cyanobacteria on an industrial scale. The proposed method avoids carrying out a drying stage after harvesting the biomass, which involves a relevant economic and energy expense at an industrial level, and uses very low amounts of solvent, mixed with water, of optimized polarity, which preferentially extract the compounds of interest, instead of all those contained in the biomass indiscriminately.

This process is robust and scalable and can be carried out in different locations, since it does not depend on a specific type of microalgae or cyanobacteria, but is adaptable to any species. In addition, it is a cost-competitive process, simple in its methodology and does not require a large infrastructure or equipment. DESCRIPTION OF THE INVENTION

As indicated above, the present invention provides an industrial-scale process for the extraction of compounds with activity that inhibits the growth of phytopathogenic microorganisms, generally referred to here as biopesticide compounds, from the biomass of microalgae and/or cyanobacteria.

Thus, a main aspect of the invention contemplates a process for obtaining, at an industrial level, a concentrated extract of biopesticide compounds from microalgae and/or cyanobacteria, comprising the following steps: i) Providing a biomass of microalgae and/or cyanobacteria,

i) Concentrate or resuspend the biomass provided in i) in a liquid medium until reaching a solid concentration of between 60 and 200 g/l, iii) Release the cellular content of the biomass obtained in i) by means of high pressure homogenization, operated at a moderate pressure, between 7 10 ⁷ and 1 ■ 10 ⁸ Pa, for an optimized time, iv) Extract the biopesticide components released in stage iii) in a stirred tank, using a mixture of an organic solvent, with a polarity between 0 and 9, and water, with a solvent concentration between 3 and 44% (v/v), at a stirring between 50 and 150 rpm, for a time between 0.5 hours and 6 hours, and at a temperature between 20°C and 65°C, and v) Separate by physical means the extract containing the biopesticide components obtained in iv).

The origin of the biomass of microalgae and/or cyanobacteria, used as a starting product in the process of the invention, is not critical, and can be obtained from its cultivation for this purpose in clean water and with chemical fertilizers, or from processes of crops in which wastewater and/or treatment of agro-industrial effluents, animal manure, digestates of organic waste, or leachate from agricultural waste are used as a nutrient source. In this context, the microalgae and/or cyanobacteria, can be produced in any type of photobioreactor, whether under autotrophic, mixotrophic or heterotrophic conditions.

The characteristics of the process of the invention have made it possible to connect the harvesting of the biomass with its processing, being able, unlike the processes described in the state of the art, to start from both wet biomass and dry biomass, since it does not require a drying stage, which represents significant energy and economic savings.

In the present invention, the expression "wet biomass" refers to the biomass that comes directly from the harvest and that is in suspension at a certain concentration. It is therefore a paste of microalgae and/or cyanobacteria made up of the biomass itself and a liquid medium, generally the aqueous medium that comes from the culture medium, although it can also be water from washing the medium, or any other liquid phase. , in variable proportions such that they confer a degree of humidity.

In a particular embodiment of the invention, the starting point is to harvest approximately between 5% and 60% of the volume of a photobioreactor that contains a variable concentration of biomass of microalgae and/or cyanobacteria in suspension between 0.1 g/l and 20g/l.

The wet biomass, coming directly from the harvest, is subjected to a concentration stage (i) to reach the required solid concentration. In a preferred embodiment, the concentration of the wet biomass is carried out by means of centrifugation or membrane filtration to the required concentration of between 60 and 200 g/L.

Optionally, the wet biomass can be further pre-concentrated by other physical methods, in particular by a sedimentation step or by flocculation, to a solid concentration of between 10 and 40 g/l. In the case of adding a flocculating agent, it is preferably selected from salts of Fe and Al, as well as cationic polyelectrolytes, in a concentration of between 0.1 and 20% (p/v).

In case of starting from dry biomass, said biomass will have to be resuspended in a liquid medium, before step iii) of cell rupture by means of high pressures, until reaching the previously indicated concentration (60-200 g/l).

The extraction of the biopesticide compounds is always done, therefore, from biomass with a solid concentration of between 60 and 200 g/L, after an optimized pre-treatment process of the biomass to achieve cell rupture of the microalgae and/or cyanobacteria.

The authors of the invention have developed a breaking process by means of high pressure homogenization, but operated at moderate pressure, for an optimized time. This process is based on a system in which the flow of wet biomass enters a chamber, where it is subjected to pressures between 7 10 ⁷ and 1 ■ 10 ⁸ Pa, said pressure causing the rupture of the cells. The time of this process can be optimized since the same fluid consisting of the wet biomass can pass through the chamber once or several times, if necessary, preferably contemplating in the present invention between 1 and 3 passes through the chamber. This type of process is industrial and is applied in other fields, but never in laboratory-scale methods, such as those described in the literature, which use ball or mortar mills (since they require starting from dry biomass) and are not industrial methods. .

Stage iv), for the extraction of the biopesticide compounds, is carried out in a stirring and heating system, such as a heated stirred tank reactor. As extracting agent, an organic solvent completely miscible with water (polarity between 0-9) is used. In preferred embodiments, the organic solvent used in iv) has a polarity index between 3.9 and 7.2, and is selected from the group consisting of acetone, acetonitrile, ethylene glycol, isopropanol, methanol, methyl ethyl ketone, nitromethane or tetrahydrofuran or combinations thereof. Step v) of separating the extract from the residual biomass of microalgae and/or cyanobacteria can be carried out by any suitable physical separation method, for example, by centrifugation or filtration.

The main advantages derived from the process of the present invention are:

It is applied to wet biomass without the need to be dried, since the method allows harvesting to be connected to biomass processing. Most, if not all, laboratory methods use dry biomass as a starting point. Biomass drying is costly and unsustainable at an industrial level, in addition to damaging some compounds of interest. However, the process of the invention can also be carried out starting from dry biomass.

Biocompatible solvents, such as acetone, are used so that the process is feasible at an industrial level and complies with current regulations. Analytical methods can use this or other non-biocompatible solvents, some of them can be toxic or explosive when handled in large quantities, so their industrial use is not allowed.

The extraction is done after an optimized biomass pre-treatment process.

The proportion of solvent, time and extraction conditions have been optimized to make it feasible and adjusted in cost at an industrial level. In analytical methods, the proportion of solvent is always in great excess, as well as the extraction time to guarantee the complete extraction of the compounds of interest. The extraction conditions are also "extreme" to prioritize the extraction of the target compounds regardless of the cost that this entails or the deterioration that it produces in the rest of the biomass.

The final product does not require solvent separation. Analytical methods or other patents require separating the solvent so that the final product is not toxic or detrimental to its application. In another main aspect of the invention, the extract obtained from the process of the invention is contemplated. The product thus obtained is a liquid biopesticide extract rich in compounds with biopesticide activity of natural origin.

The biopesticide compounds of interest are preferably selected from fatty acids, including lauric acid (C12:0), palmitic acid (C16:0), linolenic acid (18:3 n-3), oleic acid (C18:1) and fatty acid. stearic acid (C18:0), which have potential uses as antibiotics and as antifungal agents; also terpenes (some halogenated), with bactericidal, antioxidant, antimalarial, insecticidal and cytotoxic properties, polyphenols, polysaccharides, pigments such as chlorophyll and carotenoids, organic acids such as propane and butanoic acid, and other compounds such as Laxaficin A, Laxaficin B, Anatoxin -A, Eremophilone, Ambigol A, Ambigol B, Ambigin isonitrile, Pahayokolides, Microcystin and Cryptophycin).

Another main aspect of the invention contemplates a formulation with biopesticide activity that comprises the extract obtained by the method of the invention.

In a particular embodiment, the formulation comprises a dilution of the extract between 10% and 40%, a wetting compound, and demineralized water up to the final volume of the formulation.

The wetting agent used in the formulation of the final product can be selected from propylene glycol, sodium lauryl ether sulfate and ethoxylated alcohol, or any other analogous agent with similar physical-chemical properties. Preferably, ethoxylated alcohol is used at a concentration of between 2 and 20%.

Finally, another main aspect of the invention contemplates the use of the extract obtained as a biopesticide in agriculture, preferably by foliar application or fertigation in crops, using the extract in diluted form. at a concentration between 0.2 g/l and 6.0 g/l.

Next, the invention is described based on an example of its embodiment.

EXAMPLE

From the microalgae and/or cyanobacteria biomass production systems, a microalgae and cyanobacteria biomass culture was harvested in continuous mode, containing the biomass at an approximate concentration of 1 g/L, concentrating the biomass continuously until 140 g/L through a system of centrifugal nozzles in series. Next, the concentrated biomass was passed to a high-pressure homogenization system where the biomass was broken down at a moderate working pressure of 1 -10 ⁸ Pascals. Subsequently, the concentrated and homogenized biomass was transferred to a 500 L stirred tank reactor where the extraction process took place in semi-continuous mode, under controlled temperature conditions, using adequate volumes of biocompatible solvents.

In this example, acetone was used as extracting solvent at a concentration of 10 ml per g equivalent of biomass. The extraction process was carried out adjusting the agitation to 50 rpm, at a temperature of 60°C, keeping it agitated for 0.6 hours.

Subsequently, the residual biomass of the extract containing the compounds with biopesticide activity was separated. For this, the extraction volume was subjected to continuous centrifugation using a chamber and disc centrifuge. The extraction process in semi-continuous mode was repeated in batches until the total processing of the available concentrated and homogenized biomass.

From the extract concentrated in biopesticide compounds obtained, the final product was formulated. The final formulation was obtained by diluting the 12% of the extract obtained, and ethoxylated alcohol as wetting compound, at a concentration of 2%, completing with demineralized water up to the final volume.

Claims

1. Procedure for obtaining, at an industrial level, a concentrated extract of biopesticide compounds, from microalgae and/or cyanobacteria, comprising the following steps: i) Providing a biomass of microalgae and/or cyanobacteria,

i) Concentrate or resuspend the biomass provided in i) in a liquid medium until reaching a solid concentration between 60 and 200 g/L, iii) Release the cellular content of the biomass obtained in i) by means of high pressure homogenization, operated at a moderate pressure, between 7 10 ⁷ and 1 ■ 10 ⁸ Pa, for an optimized time, iv) Extract the biopesticide components released in stage iii) in a stirred tank, using a mixture of an organic solvent, with a polarity between 0 and 9, and water, with a solvent concentration between 3 and 44% (v/v), at a stirring between 50 and 150 rpm, for a time between 0.5 hours and 6 hours, and at a temperature between 20°C and 65°C, and v) Separate by physical means the extract containing the biopesticide components obtained in iv).

2. Process according to claim 1, wherein the biomass provided in step i) is wet biomass.

3. Process according to claim 2, wherein the concentration of the wet biomass in step i) is carried out by means of centrifugation or membrane filtration.

4. Process according to claim 2 or 3, where additionally the wet biomass is pre-concentrated by means of sedimentation or flocculation.

5. Process according to claim 4, wherein the wet biomass is pre-concentrated by sedimentation to a solid concentration of between 10 and 40 g/l.

6. Process according to claim 4, wherein the wet biomass is pre-concentrated by adding a flocculating agent at a concentration of between 0.1 and 20% (w/v).

7. Process according to any of the preceding claims, wherein the organic solvent used in iv) has a polarity index between 3.9 and 7.2.

8. Process according to claim 7, wherein the solvent is selected from the group consisting of acetone, acetonitrile, ethylene glycol, isopropanol, methanol, methyl ethyl ketone, nitromethane or tetrahydrofuran or combinations thereof.

9. Concentrated extract with biopesticide activity obtained according to the procedure of claims 1-8.

10. Formulation with biopesticide activity comprising the extract of claim 9.

11. Formulation, according to claim 10, comprising a dilution of the extract between 10% and 40%, a wetting compound, and demineralized water to complete the final volume.

12. Formulation according to claim 11, where the wetting agent used is ethoxylated alcohol at a concentration between 2 and 20%.

13. Use of an extract, according to claim 9, or a formulation, according to claim 10, as a biopesticide in agriculture.